PROJECT SUMMARY The parent project, ?Advancing the Understanding of Postoperative Delirium Mechanisms via Multi-Omics? (R01AG051658), uses metabolomics and lipidomics to elucidate delirium pathophysiology and identify biomarkers to enhance prediction and monitoring of delirium. This project leverages plasma samples from the NIA-funded program project, SAGES (Successful Aging after Elective Surgery) study which enrolled 460 elective orthopedic surgery patients. This Administrative Supplement, which is within the scope of the parent RO1, takes advantage of the opportunity to significantly enhance our metabolomics and lipidomics data acquisition and analysis based on recent methodological advances that improve mass spectrometric reliability, reproducibility, and consistency. As the field of biomarker discovery in large-scale mass spectrometric metabolomics and lipidomics studies has matured, criteria for accurate and reproducible quantitation has become more rigorous. It is critical to correct for measurement variation within a set of samples run on one day (intra-assay) and over time (inter-assay). This is a particular notorious problem in mass spectrometry where intra-assay and inter-assay coefficients of variation are relatively high. Our initial lipidomics and metabolomics analyses of SAGES plasma samples indeed show within batch and between batch variability, creating major challenges for meaningful data interpretation. The field of metabolomics and lipidomics has steadily advanced since our Parent RO1 application and has adopted several new best practice methods such as many pooled quality control (QC) samples, heavy isotope spike-ins and a strictly defined run order of QC versus test samples to address these challenges. The proposed Supplement Aims will adopt these rigorous standards in our metabolomics and lipidomics experiments. Supplement Aim 1. To improve and optimize the metabolomics and lipidomics workflow for discovery and validation of delirium biomarkers in plasma from the SAGES study and CSF from the HIPOR study Supplement Aim 2. To develop an optimized bioinformatics analysis workflow for metabolomics and lipidomics IMPACT: This proposal requests funds to enable incorporation of state-of-the-art methodologies into our metabolite and lipid biomarker discovery studies proposed in Parent Aims 2 and 3. Significant effort over the last couple of years has focused on improving inter-assay and intra-assay reproducibility and consistency which has led to significant advances and modifications of the previous workflows. This is now considered a gold standard for metabolomics and lipidomics analysis, but with significant additional costs. Implementation of this novel metabolomics and lipidomics workflow will have a major impact on discovery of the most accurate and reliable metabolite and lipid biomarkers for delirium in plasma and CSF. The proposed supplementary aims are well aligned with the top priority efforts of the NIH to enhance rigor and reproducibility in scientific research. This work has the potential to be high impact by maximizing biomarker yield of our Aims 2 and 3, thereby improving the resulting biomarker signatures, and leading to a better understanding of the pathophysiology of delirium.